Amplicon Sequencing Workflow: A Step-by-Step Guide

Amplicon Sequencing Process

Step 1: Sample Preparation

The first step in the amplicon sequencing workflow is sample preparation, which involves isolating nucleic acids (DNA or RNA) from the sample of interest. This step is critical for obtaining high-quality genetic material needed for downstream processes. Depending on the source of the sample—whether it be human tissue, pathogens, or environmental samples—different extraction methods may be used to optimize yield and purity. For instance, tissue samples often require mechanical or enzymatic disruption to release nucleic acids, while pathogen samples may need specialized lysis techniques to break down cell walls or viral envelopes.

In cases where the starting material is limited, low-input extraction protocols can be applied to ensure that sufficient DNA or RNA is available for amplification. Ensuring that the isolated nucleic acids are free from contaminants such as proteins, lipids, or residual chemicals is essential, as these can interfere with the subsequent library preparation process and compromise sequencing results.

Step 2: Library Preparation

Library preparation is one of the most important steps in the amplicon sequencing process, as it ensures the DNA fragments are ready for sequencing. Once the nucleic acids are isolated, specific regions of interest are amplified using polymerase chain reaction (PCR) primers. This step produces amplicons, which are the DNA fragments amplified during the PCR process. After PCR amplification, the amplicons need to be cleaned to remove unwanted primer dimers and other non-specific products. This is where advanced methods like CleanPlex technology from Paragon Genomics come in. CleanPlex uses an innovative enzymatic cleaning step to remove background noise.

Following the cleaning step, sequencing adapters are added through an index PCR step. These adapters enable the sequencing platforms to recognize the samples and allow for sample pooling, reducing sequencing costs. Additionally, the workflow includes steps like bead purification to ensure high-quality libraries. With CleanPlex technology, the entire library preparation workflow can be completed in as little as three hours, making it both time-efficient and scalable.

Step 3: Sequencing

Once the library is ready, it can be loaded onto a next-generation sequencing (NGS) platform for high-throughput sequencing. Sequencing platforms such as Illumina, Ion Torrent, or long-read instruments like PacBio or Oxford Nanopore read the amplified DNA sequences, generating large amounts of data for analysis. Amplicon sequencing is defined as a targeted sequencing method, focusing on specific genomic regions of interest.

Compared to whole genome sequencing, which examines the entire genome, targeted amplicon sequencing allows researchers to focus on specific regions, making it faster, cheaper, and more efficient. This method is particularly useful for detecting genetic variants, such as single nucleotide polymorphisms (SNPs), insertions, deletions, and copy number variations.

Step 4: Data Analysis

The final step in the amplicon sequencing workflow is data analysis. After sequencing, the raw data generated by the sequencing platforms must be processed and analyzed to identify the genetic variants of interest. This typically involves aligning the sequencing reads to a reference genome and comparing amplicon sequences to detect variations.

Data analysis software tools can also identify evolutionary relationships between organisms, perform fungal identification through analysis of the 16S rRNA gene, or detect pathogen sequences in clinical samples. One of the main benefits of amplicon sequencing is its high sensitivity, which allows researchers to detect rare mutations and variations in complex samples.

For data analysis to be effective, it’s essential to use high-quality libraries and sequencing data. Paragon Genomics’ CleanPlex technology, with its ability to reduce background noise and improve library purity, enhances the accuracy of data analysis by ensuring that the sequencing results are as clean as possible.

Benefits of Amplicon Sequencing

Amplicon sequencing offers several key benefits, making it a widely used method for targeted sequencing. These include:

• Cost-efficiency: With fewer reagents and lower sequencing costs than whole genome sequencing, amplicon sequencing is ideal for projects that require high throughput sequencing on a budget.
• Precision: By focusing on specific genomic regions, amplicon sequencing delivers highly accurate results, making it a preferred method for applications like genetic testing and pathogen detection.
• Scalability: Technologies like Paragon Genomics’ CleanPlex allow for scaling up the number of targets in a single panel, enabling the analysis of large numbers of genetic variants without sacrificing performance.
• Quick Turnaround Time: With a fast workflow, including minimal hands-on time, amplicon sequencing can provide rapid results, which is critical for applications like clinical diagnostics.

Advancing Genomic Research with Paragon Genomics

The amplicon sequencing workflow, from sample preparation to data analysis, has become a vital tool for targeted sequencing across multiple fields. Its focus on specific genomic regions, ability to detect rare variants, and cost-effective approach make it invaluable for applications like clinical diagnostics and genetic research. With advanced technologies like Paragon Genomics’ CleanPlex, researchers can achieve highly accurate results with streamlined workflows and minimal hands-on time.

Paragon Genomics continues to push the boundaries of what’s possible in the sequencing space, delivering innovative solutions that meet the evolving needs of modern genomics research. Whether for precision oncology, pathogen detection, or beyond, our technology provides the efficiency and reliability that today’s researchers rely on.